Calcium, phosphate and cholecalciferol (CC) or vitamin D3 metabolism appear tightly linked to each other through the relationship of the renal enzymes, 25-hydroxycholecalciferol-24-hydroxylase and 25-hydroxycholecalciferol-1 -hydroxylase. This enzymatic regulation in renal failure is apparently disrupted. Both modulatory effects and control of absolute levels of these enzymes have been observed. It is the purpose of this study to examine the control of these hydroxylases and to study the effect of uremic sera on them in isolated cell systems. Serially cultured cell lines derived from kidney tissue and freshly isolated cells (primary cultures) from avian and mammalian kidneys will be grown in monolayer cultures. After sufficient growth has been achieved these cells will be treated with physiologic factors (i.e., parathyroid hormome, calcitonin, varying ca2 ion and PO43-concentrations) and chemical agents (i.e., aminophylline, actinomycin D, cycloheximide) added to the medium to alter 25-hydroxycholecalciferol (25-OH-CC) metabolism by the 25-OH-CC hydroxylases. The intracellular-mechanism by which these enzyme activities are altered will be investigated. The effects of uremia on 25-OH-CC metabolism will be investigated in vitro by treating cell cultures with uremic serum (HUS). Initial studies indicate that a substance altering 25-OH-CC metabolism exists in the uremic patient. The isolation and characterization of this substance will be attempted by chromatographic, enzymatic and electrophoretic means. The effects of this material on 25-OH-CC metabolism will also be characterized.